Mechanism of multi-site phosphorylation from a ROCK-I:RhoE complex structure.
The ROCK-I serine/threonine protein kinase mediates the effects of RhoA to promote the formation of actin stress fibres and integrin-based focal adhesions. ROCK-I phosphorylates the unconventional G-protein RhoE on multiple N- and C-terminal sites. These phosphorylation events stabilise RhoE, which functions to antagonise RhoA-induced stress fibre assembly. Here, we provide a molecular explanation for multi-site phosphorylation of RhoE from the crystal structure of RhoE in complex with the ROCK-I kinase domain. RhoE interacts with the C-lobe alphaG helix of ROCK-I by means of a novel binding site remote from its effector region, positioning its N and C termini proximal to the ROCK-I catalytic site. Disruption of the ROCK-I:RhoE interface abolishes RhoE phosphorylation, but has no effect on the ability of RhoE to disassemble stress fibres. In contrast, mutation of the RhoE effector region attenuates RhoE-mediated disruption of the actin cytoskeleton, indicating that RhoE exerts its inhibitory effects on ROCK-I through protein(s) binding to its effector region. We propose that ROCK-I phosphorylation of RhoE forms part of a feedback loop to regulate RhoA signalling.
Komander
David
D
Section of Structural Biology, The Institute of Cancer Research, Chester Beatty Laboratories, London, UK.
Garg
Ritu
R
Wan
Paul T C
PT
Ridley
Anne J
AJ
Barford
David
D
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D018360:Crystallography, X-Ray; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D020125:Mutation, Missense; D010766:Phosphorylation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D020836:Protein Structure, Quaternary; D016415:Sequence Alignment; D020741:rho GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000737:chemistry* / Q000378:metabolism*
D050505:Mutant Proteins; D054460:rho-Associated Kinases; C508705:RND3 protein, human; D020741:rho GTP-Binding Proteins